The base structure of all living organisms is the cell which is structurally defined by its membranous lipoprotein envelope. The membranous network that holds the cell together maintains the ionic balance and provides the receptors for hormones and neurotransmitters that enable a cell to interact with its environment. This is pertinent to interaction with neighboring cells which enable isolated cells, tissues, or whole organisms to survive as both independent units and as participants in cellular interactions, in vitro and in vivo.
External factors which govern cell function, renewal, reproduction and death act via their effects on the phospholipid bilayer and proteins of the cell membrane. This controls the receptor-mediated signals and ionic fluxes which govern cell responsiveness and survival. Damage to the cell membrane with particular emphasis on lipid peroxidation, membrane oxidation and the action of phospholipases, affects resistance to injury, repair and host responses to environmental change and ionic and osmotic integrity.
Pathological events in a host under clinical circumstances can result in cellular insult, leading to loss of membrane integrity. The events are mediated by factors which digest and destroy cell membrane and propagate an injury by producing a cascade of cell membrane changes. By interfering with the cascade of external and internal events involving membrane integrity and toxic changes which lead to cell death, injury can be prevented, modified or reversed. This has been a major role of anti-inflammatory agents in the past.
The most important presently used clinically effective anti-inflammatory drugs include the corticosteroids and the non-steroidal anti-inflammatory drugs (NSAIDs). Corticosteroids inhibit the activity of cell phospholipases among other actions. NSAIDs inhibit the metabolism by cyclooxygenase of arachidonic acid released by phospholipases. These drugs act to control inflammation and to minimize cell injury by regulating the breakdown of phospholipids. These drugs also affect the action of the products of phospholipid breakdown leading to the formation of prostaglandins and leukotrienes which are produced in increased quantities in inflammation and promote cell dysfunction and injury.
In addition, cellular and extracellular phospholipases may be activated by the generation of oxygen free radicals. This can establish a damaging cycle as phospholipase activation can release free radicals which, in turn, activate more phospholipases. In this regard, free radicals are produced from the fatty acids which are released by the action of phospholipases and then converted to prostaglandins and leukotrienes by cyclooxygenase and lipoxygenase enzymes with oxygen free radical production as a by product. Fatty acids and free radicals are known to be prime mediators in the cascade of reactions that result in membrane injury, cell death and inflammation. Phospholipase A.sub.2 (PLA.sub.2), a key enzyme in the metabolism of phospholipid, can promote fatty acid release. PLA.sub.2 may be activated by a variety of factors involving hormonal, neural, metabolic, or immunologic pathways.
One of the hallmarks of inflammation and cell injury is the breakdown of cellular membrane phospholipid. Phospholipids are the major structural building blocks of the cell membrane; they give rise to the barrier-structural and functional properties of membranes and their integrity is crucial to normal cell responsiveness and function. Phospholipid changes in cell membrane integrity, particularly changes in fatty acids at the 2 position, alter the fluidity of cell membranes, cell receptor function and the availability of cellular contents to the external environment. The breakdown of phospholipid membranes results in lysis of cells, produces holes in the cell membrane, affects ion channels and membrane receptors which destroy cellular integrity and functional responses.
During inflammation, phospholipases, from whatever source, that are normally under the control of natural suppressor systems, are activated to degrade membrane phospholipid which, in turn, generates oxygen free radicals. PLA.sub.2 is a key enzyme which is activated in inflammation to metabolize substrate phospholipids and release free fatty acids. These fatty acids (i.e., arachidonate) released by PLA.sub.2 are converted to potent biologically active metabolites, lysophospholipids, prostaglandins, and leukotrienes. These are themselves substrates for other enzymes leading to the production of thromboxanes, platelet activating factor and other substances, with the concomitant generation of oxygen free radicals.
Phospholipases, particularly PLA.sub.2, as membrane targeted enzymes, play an important role since expression of their activity results in further production of inflammatory mediators leading to membrane injury which propagates damage within the cell itself or to adjacent tissue. Thus, the spread of injury from the initial site to contiguous or distant sites can be promoted by the activation and/or release of PLA.sub.2.
In addition to the intrinsic membrane-related tissue breakdown via the activation of PLA.sub.2, phospholipases, and particularly PLA.sub.2, are part of the normal defense system of the body. PLA.sub.2 is found in human white blood cells (WBCs). WBCs play a role in resisting infection, but when these cells are mobilized to ward off injury and infection, PLA.sub.2 is released from adherent and circulating WBCs and produces local tissue activation which can increase the extent of initial injury. In addition, WBCs adhere to blood vessel walls where they release enzymes such as PLA.sub.2. WBCs also generate free radicals such as superoxide, in large quantities, and thus promote damage to the vascular endothelium, lung alveoli or to tissue sites contiguous with WBC infiltration or concentration. Where inflammation is found, WBCs are usually present in abundance and the WBCs adhere to vascular endothelium, with subsequent release and activation of PLA.sub.2 resulting in damage to vascular integrity during shock and ischemia. Thus, in spite of being a prime defense system of the body against infection, WBCs can also damage the body by propagating injury and inflammation.
A classical description of inflammation is redness and swelling with heat and pain. Inflammation has been defined as the reaction of irritated and damaged tissues which still retain vitality. Inflammation is a process which, at one level, can proceed to cell death, tissue necrosis and scarring. At another level, inflammation can be resolved with a return to normalcy and no apparent injury or with minimal changes, i.e., pigmentation, fibrosis or tissue thickening with collagen formation related to healing and scarring.
Microscopically, inflammation has been described as: (1) atony of the muscle coat of the blood vessel wall; (2) endothelial adherence of inflammatory cells followed by migration of these cells from the vascular space into tissue.
The events described above are often mediated by phospholipase activation, followed by fatty acid release and the formation of free radicals. Cytokines, secreted by immune cells, induce PLA.sub.2 secretion by their actions on a variety of cells. Interleukin-6 stimulates hepatocytes to increase PLA.sub.2 secretion many-fold. Interleukin-1 and tumor necrosis factor induce PLA.sub.2 secretion by endothelial cells and by chondrocytes. Thus, immune cell products directly stimulate the hydrolysis of membrane phospholipids and production of arachidonic acid metabolites by a variety of target cells, amplifying the inflammatory response.
Alternatively, increased phospholipase activity can relate to exogenous enzyme released from infecting pathologic organisms such as viruses, bacteria, Rickettsia, protozoa, and fungi. These pathogens often possess phospholipases as factors intrinsic to their infectious activity. In the case of Naegleria fowleri, a pathogenic amoeba with affinity for the brain, destruction of brain membranes induced by phospholipases secreted by Naegleria can occur at sites in the brain distant from where the organism is localized. In another example toxoplasma cannot enter the host cell if its PLA.sub.2 enzyme is inhibited by a specific drug. What is needed to treat certain infections, particularly intracellular pathogens, is an effective PLA.sub.2 inhibitor. Such an effective PLA.sub.2 inhibitor is particularly needed in cases of protozoal infections for which there are few effective antibiotics.
PLA.sub.2 is also one of the major toxic components of snake venom. Bites of certain snakes inject venom containing PLA.sub.2 into the wound, causing toxic and inflammatory responses which may be lethal. What is needed are inhibitors of PLA.sub.2 which may be administered to recipients of snake bites and bites of other animals.
Pathologic effects of phospholipases may be local, regional or systemic. These pathologic effects are governed by the phospholipase enzyme released, the level of albumin, natural inhibitors of enzyme action, and factors of diffusion, circulation and tissue vulnerability based on intrinsic inhibitors or the susceptibility of previously damaged or oxidized membranes or proteins to phospholipase action.
Inflammation is associated with trauma, infection and host defense reactions related to direct bacterial or virus killing by the associated immune responses. In general, immune responses can be both beneficial, protective or tissue damaging as can be seen in their role to promote resistance to infection or cure of infection. Alternatively, immune responses may produce autoimmune phenomena that result in allergy, i.e., asthma, urticaria, in graft versus host disease, in glomerular nephritis, in rheumatic fever, or in lupus and rheumatoid arthritis.
In regard to the current treatment of inflammation, corticosteroids are effective anti-inflammatory agents, but must be used cautiously because they are powerful immunosuppressants and inhibitors of fibroblastic activity necessary for wound healing and bone repair. In addition, corticosteroids have powerful hormonal activities and their toxic side effects involve interference with wound repair and bone matrix formation, sodium retention, potassium loss, bone demineralization, decreased resistance to infection, and diabetes. Corticosteroids also have effects on steroid formation, cataracts, blood pressure, protein utilization, fat distribution, hair growth and body habitus. Alternatively, the clinically active NSAIDs, such as aspirin, indomethacin, ibuprofen, etc., work by inhibiting the conversion of free fatty acids to prostaglandins. The side effects of NSAIDs include gastric ulceration, kidney dysfunction and Reye's Syndrome. Metabolites of prostaglandin can be either damaging or protective to cells depending on the structure of the prostaglandin produced or utilized pharmacologically and the route of administration, cell or tissue effected.
As discussed previously, in conjunction with fatty acid release, leukotrienes are generated as part of phospholipid cell membrane mediated injury produced by phospholipase activation. These leukotrienes produced from membrane phospholipid breakdown damage tissue through direct toxic action, effects on ionic channels, and associated free radical formation. Leukotrienes also damage tissue by indirect effects on vascular smooth muscle or on the vascular endothelial lining via effects on platelets, WBCs, or endothelial cells, or secondarily through effects on constriction of smooth muscle. Leukotrienes are responsible for smooth muscle constriction leading to bronchospasm and the asthmatic attacks seen in allergy or infectious asthma. Thus, there is an ongoing search for leukotriene inhibitors for clinical application in the treatment of allergy, asthma and tissue injury and inflammation.
Because the phospholipase-activated biochemical pathway for the formation of prostaglandins and leukotrienes derived from free fatty acids is branched, inhibition of one branch of this pathway, as with NSAIDs, can create an imbalance in these reactive metabolites. This imbalance may actually aggravate inflammation and promote cell injury as evidenced by the gastric ulcerative side effects of NSAIDs.
Due to these adverse effects of both steroids and NSAIDs, there is great clinical interest in identifying phospholipase inhibiting agents that do not have steroidal or NSAID side effects, but like corticosteroids modulate the first step leading to the production of injurious metabolites, fatty acids and free radicals.
Free radicals, produced by white blood cells, tissue injury or metabolic processes, are highly reactive chemical species which, in the case of tissue injury, are most often derived from respiratory oxygen. Oxygen, while necessary for energetics of life, is also a toxin which, as the chemically related superoxide, or as peroxides, can damage tissue instead of supporting it. Free radicals derived from oxygen are critical to damage produced by radiation, inflammation, reperfusion tissue injury or through excess oxygen inhalation or exposure. Free radicals are used by white blood cells to destroy infecting organisms, but can, under circumstances of shock, infection and ischemia, damage or destroy the tissue they were meant to protect. Free radicals, induced by radiation, oxygen exposure, chemical agents (i.e., alkylating agents, dioxin, paraquat) or white blood cell reactions may damage tissue or be involved in mutational changes associated with aging, radiation or chemotherapy injury, the development of cancer, and hyperimmune proliferative disease such as rheumatoid arthritis. In addition, these reactive chemical species can, through oxidation of proteins, enhance the vulnerability of proteins to protease digestion.
The exact pathologic mechanisms of many skin inflammations, such as atopic dermatitis, are not clear, but probably involve inflammatory cells which can secrete or respond to PLA.sub.2. Allergic diseases involve tissue mast cells, which can be primed or triggered by PLA.sub.2 for the release of their inflammatory granule contents, such as histamine. These cells also release additional PLA.sub.2. What is needed are inhibitors of PLA2 that adequately penetrate skin after topical application and possess prolonged anti-PLA.sub.2 activity.
Previous published studies have demonstrated high levels of a proinflammatory PLA.sub.2 in human herniated vertebral discs. The isolated enzyme is toxic to dorsal root ganglion cells in culture and excised sciatic nerve. While not wanting to be bound by this statement, it is believed that PLA.sub.2 may mediate inflammation and nerve tissue damage in spinal cord injury and in sciatic nerve inflammation and may also mediate a variety of neurological inflammatory conditions. Recently, Stephenson et al., (Neurobiology of Disease 3:51-63 (1996) have observed elevated cytosolic PLA.sub.2 activity in brains with Alzheimer's disease.
PLA.sub.2 also has the capability to induce severe, delayed neurotoxicity syndrome, including extensive cortical and subcortical injury to forebrain neurons and fiber pathways, when injected intracerebroventricularly as described by Clapp et al. (Brain Research 693:101-111,1995) the entirety of which is incorporated herein by reference. We have also observed that preparations of PLA.sub.2 and homogenates of human vertebral disks containing extracts of the nucleus pulposus are inflammatory when injected into the mouse paw and induce edema. Edema induced by human disk homogenate is maximal between 1-3 hrs and remains so for at least 6 hrs. These results support the hypothesis that leakage of nucleus pulposus from a herniated disk may promote inflammation in human disk disease. Accordingly, what is needed are inhibitors of PLA.sub.2 mediated inflammatory processes. Such inhibitors should alleviate the inflammation and resultant pain and discomfort associated with disk disease and other neurological inflammatory conditions.
Tissues that are excised from animals for subsequent transplantation into recipients often display damage following transplantation during reperfusion of ischemic tissue. Both ischemia and reperfusion increase PLA.sub.2 activity and release leading to inflammatory processes with marked activation of the vascular endothelium. These processes decrease the probability of successful transplantation thereby increasing the incidence of rejection and the need for additional immunosuppressive therapy. Such problems greatly increase morbidity and mortality, increase the costs of treatment and insurance, and result in lost time at work. What is needed are drugs that will inhibit PLA.sub.2 activity and enhance tissue preservation before transplantation thereby decreasing ischemia reperfusion injury.
Infections caused by parasites constitute a major public health problem throughout the world for humans and animals, annually resulting in significant incidence of disease, suffering and death. Parasites such as those that cause malaria and other protozoal parasites of animals and humans are especially troublesome. We have found that the PLA.sub.2 inhibitor, quinacrine (mepacrine), significantly reduced molting of larval forms of an animal filarid. What is needed are new compounds that effectively inhibit PLA.sub.2 activity for application to parasites such as those causing malaria and other protozoal parasites injurious to animals and humans.
A previous study by Clay et al. (Third International Congress: Eicosanoids & Other Bioactive Lipids in Cancer, Inflammation, & Radiation Repair, Abstract #162) reported that the product of PLA.sub.2 activation, 1-acyl lysophospholipid, which affects membrane fluidity, accumulates in stored blood and may be taken up by white blood cells (WBCs) and used to make platelet activating factor (PAF) thereby "priming" WBCs during storage and promoting injury during subsequent transfusion. It has been suggested that increased PLA.sub.2 activity may perturb cells in storage. What is needed are compounds that protect blood cells and other cells during storage so that these cells will not cause problems when utilized.
Accordingly, what is needed are compounds and methods of using these compounds which provide protection against the deleterious effects of PLA.sub.2 activation. These compounds should be capable of inhibiting PLA.sub.2, thereby decreasing the PLA.sub.2 metabolites which are substrates for the cyclooxygenase, 5-lipoxygenase, 12-lipoxygenase, and other enzymatic pathways which lead to formation of cyclic endooperoxides, prostaglandins (such as prostacyclin and thromboxane), leukotrienes, and platelet activating factor. These compounds should decrease inflammatory processes and free radical production in a variety of tissues and cells. They should be capable of being administered in vivo (topically, orally, by injection and through other means), ex vivo and in vitro and should also exhibit low or no toxicity. These compounds should display different solubilities in lipid and aqueous systems depending on the mode of application and the desired target.